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Supplemental material for Miller et al. 2020

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posted on 12.10.2020 by Danny Miller, Lily Kahsai, Kasun Buddika, Michael J. Dixon, Bernard Kim, Brian R. Calvi, Nicholas Sokol, Scott Hawley, Kevin Cook
Table S1. Reagent table
Table S2. Genomic coordinates of third chromosome balancer breakpoints
Table S3. Breakpoint complementation crosses
Table S4. Control crosses for breakpoint complementation tests
Table S5. Loss of irradiation-induced follicle cell apoptosis from the 94D TM3 breakpoint disrupting p53
Table S6. Complementation tests for anti-HRP immunodetection
Table S7. Missense, stop-gained and splice-site variants caused by SNPs or indels shared among all eight FM7 chromosomes.
Table S8. Missense, stop-gained and splice site variants caused by SNPs or indels shared among all three FM7a chromosomes.
Table S9. Missense mutations stop-gained, and splice site variants caused by SNPs or indels shared among all five FM7c chromosomes.
Table S10. Missense, stop-gained and splice-site variants caused by SNPs or indels shared by at least 15 of 18 TM3 chromosomes.
Table S11. Missense, stop-gained and splice-site variants caused by SNPs or indels unique among all FM7a, FM7c or TM3 chromosomes.
Table S12. Mapping the Ank2 mutation present on a subset of TM3 chromosomes
Table S13. Complementation tests to characterize the female-sterile mutation on CyO from the Df(2L)bhe, In(2R)bhe, Ir21abhe CG3164bhe/CyO stock
File S1. Sequence within the In(2L)Cy and In(3R)C inversion breakpoints.

Figure S1. Specific intestinal epithelial cells are identified by anti-HRP antibodies. The mira-His2A.mCherry.HA construct expresses mCherry- and hemagglutinin-tagged Histone 2A under the control of miranda regulatory sequences. Panels A and B show that mira-His2A.mCherry.HA is expressed in progenitor cells (stem cells and enteroblasts) and enteroendocrine cells. mCherry was detected with anti-RFP antibodies (red), progenitor cells were marked by UAS-Stinger expression (green) under the control of the progenitor cell-specific P{GawB}NP5130 driver (esg-GAL4), enteroendocrine cells were detected (gray) with antibodies against Prospero (Pros), and all nuclei were stained with DAPI (blue). Panel A shows representative images of mira-His2A.mCherry.HA expression in the five primary midgut regions (R1–R5). Panel B shows a specific region (outlined in panel A) with separated fluorescence channels. It shows that progenitor and enteroendocrine cells are clearly distinguished by the level of mira-His2A.mCherry.HA expression: mCherry is expressed strongly in GFP-expressing progenitor cells and weakly in Pros-expressing enteroendocrine cells (yellow arrowheads). Panels C and D show that almost all progenitor and enteroendocrine cells expressed mCherry (though we have noted that lower percentages of enteroendocrine cells express mCherry in other genetic backgrounds). Standard deviations are indicated for the five intestines examined. Panels A–D present data from female progeny of a cross of M{mira-His2A.mCherry.HA}ZH-2A, w*; P{GawB}NP5130/CyO females to P{UAS-Stinger}2 males. Panel E shows that anti-HRP staining is similar to mira-His2A.mCherry.HA expression: strong in progenitor cells and weak in a subset of enteroendocrine cells (yellow arrowheads). Anti-RFP antibody staining to detect mCherry is shown in red, anti-HRP staining in green, anti-Pros staining in gray, and DAPI staining of nuclei in blue. Panel E shows R5 cells from female progeny of a cross of M{mira-His2A.mCherry.HA}ZH-2A, w* females to y1 w67c23; P{lacW}esgk00606/CyO males.

Figure S2. Characterizing CyO from the Df(2L)bhe, Ir21abhe stock. In our initial crosses combining CyO from the Df(2L)bhe, Ir21abhe stock and molecularly defined deletions (Table S13), we saw that Df(2L)ED5878 failed to complement this CyO for female sterility (indicating the presence of fs(2)21Ba1), but Df(2L)ED50001 and Df(2L)ED929 complemented. The region of Df(2L)ED5878 is shown in this image from GBrowse (Thurmond et al. 2018). In follow-up crosses, we complementation tested terminal deletion chromosomes that had been characterized previously by Caggese et al. (1988) and Larsson et al. (1996). Because terminal deletion chromosomes continue to lose end sequences until telomeres are added (Biessmann and Mason 1988) and we do not know if the terminal deletions had stabilized at the time they were characterized, we can use previous endpoint localizations only as minimal estimates of deletion extents. Deletions that had previously been shown to delete net, but not Sam-S (Df(2L)PM4, PMA, PMD and net62) complemented fs(2)21Ba1. Df(2L)net18—which failed to complement the lethality of a Sam-S mutation but complemented the lethality of a mutation in CG3164—also failed to complement fs(2)21Ba1. These results place fs(2)21Ba1 in the seven-gene interval to the right of net and to the left of CG3164. Df(2L)PM44, which had previously been shown molecularly to break within Sam-S (Larsson et al. 1996), failed to complement fs(2)21Ba1. If Df(2L)PM44 was stabilized at the time its breakpoint was mapped, fs(2)21Ba1 lies in the four-gene Zir to Sam-S interval. Females carrying fs(2)21Ba1 and Sam-SR23 were sterile (rather than lethal as would be expected for strong loss-of-function genotypes), which suggests fs(2)21Ba1 is a hypomorphic Sam-S mutation, but females carrying fs(2)21Ba1 and Sam-S1 were fertile. Sam-S1 is a hypomorphic allele and intragenic complementation of hypomorphic alleles is not unusual, but further tests are needed to exclude other possibilities.  

Figure S3. Extremely distal meiotic crossovers between Ir21a and fs(2)21Ba do not generate mutation-free chromosomes in the broadhead stock. The Df(2L)bhe, Ir21abhe chromosome is lethal in combination with CG316421Ba-1 (as we are renaming l(2)21Ba1 here), CG3164MI10825 or CG3164KG08120 (Table S13). The causative mutation, CG3164bhe, is an A to T transversion at 2L:123,157 predicted to truncate the CG3164 protein eight amino acids from the C-terminus (leucine to stop). The presence of CG3164bhe on the Df(2L)bhe, Ir21abhe chromosome lying proximal to fs(2)21Ba1 on CyO adds to the stability of the Df(2L)bhe, Ir21abhe/CyO stock, because any crossovers that might occur between Ir21abhe and fs(2)21Ba1—despite telomeric suppression—cannot give rise to recombinant chromosomes lacking mutations. This is depicted generically in the figure, where crossovers that occur between distal mutations (m– and fs–) can lead to stock breakdown (A) in the absence of a second deleterious mutation (l–), but lead to recombinant chromosomes that are eliminated from the stock when the second mutation is present (B). The presence of CG3164bhe suggests that the broadhead phenotype seen in homozygotes (Nüsslein-Volhard et al. 1984; RG and Nüsslein-Volhard 1987) is not entirely attributable the Df(2L)bhe and Ir21abhe lesions. Likewise, a 5.6 Mb inversion on the Df(2L)bhe, Ir21abhe CG3164bhe chromosome (49E1;54B17, 2R:12,889,785;17,485,201), which we found through sequencing, may also contribute to the phenotype.


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Identification and Characterization of Breakpoints and Mutations on Drosophila melanogaster Balancer Chromosomes

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